Methods of treating sickle cell disease

ABSTRACT

The present invention relates to methods of treating sickle cell disease comprising reducing, in a subject in need of such treatment, the adherence between sickle RBCs and leukocytes. It is based, at least in part, on the discovery that leukocytes play a direct role in the initiation of venular occlusion. The present invention further provides for methods for identifying agents which decrease SS-RBC/leukocyte adherence and for animal models which may be used to further elucidate the mechanism of vaso-occlusion in sickle cell crises.

SPECIFICATION

[0001] The subject matter described in this provisional application wasdeveloped with support from the following grants: National Institute ofHealth (“NIH”) Grant No. P60-HL28381 to Sergio Piomelli, NIHDK Grant No.DK 56638-01 to Paul Frenette, and NIHLBI Grant No. HL 19278 to BarryColler, so that the United States Government has certain rights herein.

INTRODUCTION

[0002] The present invention relates to methods for treating sickle celldisease in which adherence between sickled erythrocytes and leukocytesis inhibited. It is based, at least in part, on the discovery that, inanimal models of sickle cell disease, sickled erythrocyte/leukocyteadhesion plays a direct role in the initiation of vaso-occlusion, thecause for sickle cell crises in humans.

BACKGROUND OF THE INVENTION

[0003] Half a century ago, Linus Pauling first showed that sickle cellanemia is a molecular disease (Pauling, 1949; for full citations seelist in Section 8, infra). It was later demonstrated that the diseaseoriginated from a missense mutation within the β-globin gene, leading tothe substitution of valine for glutamic acid on the outer surface of theglobin molecule. This amino acid substitution renders the sickle cellhemoglobin (“HbS”) less soluble and prone to polymerization upondeoxygenation (Hoffman, 2000). Erythrocytes (red blood cells, “RBC”)carrying polymerized HbS are thus less deformable and may obstructmicrovessels. This vascular occlusion, producing tissue ischemia andinfarction, represents a major cause of morbidity and mortality amongsickle cell disease patients. Despite recent therapeutic advances withthe use of hydroxyurea and butyrate (Charache, 1995; Atweh, 1999) manypatients remain severely symptomatic and thus, may benefit fromalternate therapeutic modalities.

[0004] Over the years, it has become clear that the clinicalmanifestations of sickle cell disease extend far beyond the homozygousglobin mutation. Seminal findings were the discovery that sickle (“SS”)RBCs, unlike normal RBCs, could adhere to stimulated endothelium invitro and that SS-RBCs' adhesion correlated with the clinical severityof sickle cell disease(Hoover, 1979; Hebbel, 1980 (a) and (b)).Subsequent studies have recognized the importance of plasma factors inSS-RBC adhesion to the endothelium (Wautier, 1983; Mohandas, 1984) andrevealed that the deformable “low-density” cells were more adherent thanthe dense sickle-shaped cells (Mohandas, 1985; Barbarino). Other elegantstudies by Kaul and coworkers subsequently showed using a rat mesocecumex vivo perfusion model that SS-RBCs adhered exclusively in venules(mostly small post-capillary and collecting venules) and confirmed thatadhesion was density-class dependent (light-density reticulocytes andyoung discocytes being most adherent; Kaul, 1989). Collectively, theseobservations lead to the current multistep model, shown in FIG. 1A, bywhich light-density SS-RBCs first adhere in post-capillary venules,after which secondary trapping of dense cells may produce vascularobstruction and local ischemia. These transient obstructions may induceHbS polymerization, which would increase RBC rigidity and exacerbatevascular occlusion.

[0005] Multiple adhesion molecules have been shown to participate inSS-RBC/endothelium interactions (FIG. 1B), Soluble adhesion moleculesand matrix proteins were first recognized, and may function as a bridgebetween two cellular adhesion receptors or may recruit SS-RBCs directlyto the vessel wall's matrix. These include fibrinogen and fibronectin(Wautier, 1983; Kasschau, 1996), von Willebrand factor (vWF; Wick, 1987;Kaul, 1993), laminin (Hillery, 1996; Lee, 1998) and thrombospondin(“TSP;Sugihara, 1992; Hillery, 1999). Several ” putative cellularcounter-receptors have been suggested, although many are controversialor still poorly defined. For example, studies have suggested that TSPmay interact with integrin associated protein (CD47; Gao, 1996) andsulfated glycolipids (Hillery, 1996), phosphatidylserine (Mandori, 2000)and CD36 (Sugihara, 1992) on the SS-RBC membrane. Other studies havesuggested that CD36 is not involved in TSP-mediated sickle cell adhesion(Hillery, 1996; Joneckis, 1996). Membrane damage to SS-RBC with loss ofphospholipid asymmetry (Frank, 1985) may expose phosphatidylserine aswell as sulfated glycolipids which can interact with vWF and laminin(Roberts, 1986). Membrane damage to SS-RBC might also expose a portionof band 3 which may contribute to SS-RBC's adhesion with endothelialcells (Thevenin, 1997). Basal cell adhesion molecule/Lutheran protein(B-CAM-LU), the protein that carries the Lutheran blood group, was alsoshown to be a laminin receptor in SS-RBCs (Udani, 1998; Parsons, 2001).Finally, the integrin α₄β₁, one of the first sickle RBC adhesionreceptor identified on sickle reticulocytes (Swerlick, 1993; Joneckis,1993; Gee, 1995), can interact with vascular cell adhesion molecule-1(“VCAM-1”) on activated endothelium. To date, few receptors for SS-RBCshave been identified on activated endothelium. In addition to VCAM-1(Swerlick, 1993; Gee, 1995), α₅β₃ has been proposed to play an importantrole since functional inhibition of this receptor drastically reducedSS-RBC accumulation on platelet activating factor (“PAF”)-stimulatedmicrovasculature in the ex vivo rat mesocecum (Kaul, 2000). Recent dataalso indicate that P-selectin may mediate SS-RBC adhesion to endothelialcells (Matsui, 2000). The foregoing studies of SS-RBC adhesion, however,suffer the shortcoming of having been performed in vitro or, in the caseof Kaul, 2000, ex vivo; the mechanisms of vaso-occlusion had not, priorto the present invention, been explored in vivo.

[0006] Several mouse strains expressing HbS have been generated in thelast decade. These transgenic strains have been used to study thepathophysiology of sickle cell disease in vivo, and may be divided intotwo broad categories: i) transgenic mice expressing both the endogenousmurine and human globin genes, and (ii) transgenic mice expressingexclusively human globin genes (Nagel, 1998). So-called “SAD” micerepresent one example of transgenic animal models for sickle celldisease in which the human β-globin transgene contains three naturalmutations that enhance Hb sickling: HbS, HbS-Antilles and Hb D Punjab(hence the acronym “SAD”). RBCs from SAD mice carry approximately 19%human hemoglobin. Although associated with a significant perinatalmortality (when a SAD mouse is bred with a wild-type animal, thefrequency of SAD offspring is about 30%, rather than the expected 50%),adult SAD transgenic mice are relatively healthy, suffering neitheranemia nor reticulocytosis unless exposed to hypoxemic conditions(Trudel, 1991; Trudel, 1994). Transgenic “knock-outs” (hereinafterreferred to as “sickle cell” or “SS” mice) were developed by sequentialbreeding of mice deficient in α and β globins with transgenic animalsexpressing both human α and β^(s) globins; such SS mice are geneticallyidentified as Tg(Hu-miniLCRα1^(G)γ^(A)γδβ^(S))mα−/−β−/−. These animalsdisplay a drastic phenotype characterized by severe anemia with highreticulocyte counts, splenomegaly and evidence of end-organ damage(Paszty, 1997; Ryan, 1997). Although the hematological and histologicalpictures in SS mice resemble that of patients, the phenotype in mice ismore severe and their viability is reduced. When a male SS mouse is bredwith a mouse heterozygous for β-globin expression(Tg(Hu-miniLCRα1^(G)γ^(A)γδβ^(S))mα−/−β−/+), less than 10% of theoffspring exclusively express human globins, instead of the expected50%. The reduced viability of SS mice has hampered the progression of invivo studies and the development of useful models to evaluate themechanisms of vaso-occlusion.

[0007] It had been noted, prior to the present invention, that sicklecell patients with leukocyte counts greater than 15,000/microliter havean increased risk of death (Platt, 1994), that lower neutrophil countswere associated with a lower crises rate in sickle cell patients treatedwith hydroxyurea (Churache, 1996) and that treatment with granulocytecolony stimulating factor (“G-CSF”, which increases leukocyte counts)induced a sickle cell crisis (Abboud, 1998). Schwartz, 1985, reportedincreased adherence of sickle RBCs to cultured peripheral bloodmonocytes in vitro, wherein irreversibly sickled RBCs and deoxygenatedRBCs were most adherent and adhesion appeared to correlate with theexposure of phosphatidylserine to the outer membrane leaflet. Hofstra etal., 1996, reported that, in vitro, SS-RBCs can bind activatedneutrophils in a static in vivo adhesion assay, an interaction which wasmore pronounced in the presence of autologous sickle cell plasma.Binding of SS-RBCs to activated neutrophils was partially inhibited byRGDS peptides and human IgG, suggesting than one or more integrin(s) andneutrophil Fc receptors may be involved. SS-RBC adhesion also induced anoxidative burst characterized by the production of free radicals byactivated neutrophils (Id.) Further, it had been noted thatanti-inflammatory agents such as methylprednisolone may be effective indecreasing the duration of sickle cell crisis episodes (Griffin, 1994).A recent study using a sickle cell mouse model indicated that theinflammatory response (number of adherent and emigrated leukocytes andoxidant production) resulting from hypoxia and reoxygenation wasincreased in sickle cell transgenic mice compared to control animals(Kaul, 2000).

[0008] Prior to the present invention, however, it had not beenappreciated which of the many potential aspects of the inflammatoryresponse was directly associated with vaso-occlusion.

SUMMARY OF THE INVENTION

[0009] The present invention relates to methods of treating sickle celldisease comprising reducing, in a subject in need of such treatment, theadherence between sickled RBCs and leukocytes. It is based, at least inpart, on the discovery that leukocytes play a direct role in theinitiation of venular occlusion. The present invention further providesfor methods for identifying agents which decrease SS-RBC/leukocyteadherence and for animal models which may be used to further elucidatethe mechanism of vaso-occlusion in sickle cell crises.

DESCRIPTION OF THE FIGURES

[0010] FIGS. 1A-B. Erythrocyte interactions with the vessel wall. (A)depicts the current paradigm's conception of events leading tovaso-occlusion. (B) depicts putative adhesion pathways involved in theinteractions of RBCs, endothelial cells (“ECs”) and the vascular matrix.PS=phosphatidylserine; BCAM/Lu=basal cell adhesion molecules/Lutheranprotein; FN=fibronectin; vWF=von Willebrand factor; TSP=thrombospondin;LN=laminin; VCAM-1=vascular cell adhesion molecule-1.

[0011]FIG. 2. Triton-X-100 gel electrophoresis of tail blood samples; 10micrograms of protein, determined spectrophotometrically, were loadedper lane. The first two lanes represent normal mouse and humanhemoglobin and the next five lanes represent mixtures with decreasingamounts of human hemoglobin.

[0012]FIG. 3. Diagrammatic representation of intravital microscopy(“IVM”) protocol. The two recording periods are designated IVM-1 andIVM-2.

[0013] FIGS. 4A-B. Erythrocyte/leukocyte interactions in wild-type, SAand SS-transplanted mice in vivo. (A) depicts the number ofRBC/leukocyte interactions quantitated in venules filmed between 30 and90 minutes after cremaster surgery, expressed as the number ofinteractions per minute per 100 microns of venular length. (B) showsthat the number of RBC/leukocyte interactions correlates with time aftersurgery. Each dot in the scattergraph represents a single venule.

[0014] FIGS. 5A-C. Digital stillframes obtained from intravitalmicroscopy of the cremaster microcirculation stimulated by TNF-α. (A) isan image from an inflamed venule (30 microns) from an SA-transplantedanimal showing adherent (white arrows) and rolling (white stars)leukocytes; no RBC are seen since free-flowing RBC move too rapidly tobe distinguished by this technique. Blood flow is from right to left.(B) is an image from an inflamed venule (20 microns) from anSS-transplanted mouse showing numerous RBCs (arrowheads) interactingwith adherent leukocytes (arrows). Blood flow is left to right. (C) isan image from a large venule with two adherent leukocytes in the center(arrows). One leukocyte has “captured” two RBCs (one sickle-shaped(arrowhead), the other discoid). Diagonal bars mark the vessel wall.Blood flow is from bottom to top.

[0015]FIG. 6. Shear rates in cremasteric venules before and after TNF-αadministration.

[0016] FIGS. 7A-B. Leukocyte rolling and adhesion in cremastericvenules. The numbers of rolling (A) and adherent (B) leukocytes weredetermined on video recordings from intravital microscopy experiments.n=30-44 venules from 3-5 mice; *p<0.0005, #p<0.005.

[0017] FIGS. 8A-B. P- and E-selectin deficiency protects fromvaso-occlusion. (A) is a view of two post-capillary venules (arrows) anda collecting venule from a SS-P/E−/− transplanted mouse after TNF-αstimulation. No leukocyte rolling and very little leukocyte adhesionwere observed and the blood flow (left to right) was preserved. (B)depicts shear rates before and after TNF-α administration in SS-P/E−/−transplanted mice. The wild-type recipients, shown in FIG. 6, are shownfor comparison.

DETAILED DESCRIPTION OF THE INVENTION

[0018] For clarity of disclosure, and not by way of limitation, thedetailed description of the invention is divided into the followingsubsections:

[0019] (a) methods of treating sickle cell disease;

[0020] (b) methods of identifying agents useful in treating sickle celldisease; and

[0021] (c) animal model systems.

Methods of Treating Sickle Cell Disease

[0022] The present invention provides for methods of treating sicklecell disease in which venular occlusion by sickle erythrocytes(“SS-RBCs”, which contain HbS and may be in the sickled or in a discoidconformation) adherent to leukocytes is decreased. The phrase “method oftreating” sickle cell disease is used herein to indicate decreasing theoccurrence and/or severity of any one or more of the following signs andsymptoms: pain, anemia, infection, stroke, tissue damage, visualimpairment, bone infarction, jaundice, and gall stones, and themanifestations of “sickle cell crisis”.

[0023] The methods of the present invention may intervene in the processby which SS-RBC adhere to leukocytes and initiate venular occlusion atthe point where a SS-RBC adheres to a leukocyte and/or the point atwhich a leukocyte and/or the SS-RBC/leukocyte complex binds to thevenule endothelium. Such methods may be directed at the cellular level(for example, decreasing the number of leukocytes) or may be directed atthe molecular interactions between the SS-RBC and leukocyte or betweenthe leukocyte or the SS-RBC/leukocyte complex and the endothelial cell.

[0024] The recruitment of leukocytes into inflamed tissue has been wellcharacterized at the molecular level. It is now recognized thatleukocyte extravasation represents a multi-step process initiated byleukocyte tethering and rolling along the vessel wall of post-capillaryvenules. The tethering and rolling steps are largely mediated byselectins and their ligands. Rolling on selectins and their ligandsallows leukocytes to interact with chemokines on the surface of theactivated endothelium. These chemokines may activate the leukocyte andchange the conformation of β₂ integrins into a high-affinity state,allowing firm adhesion and subsequent diapedesis via the interactions ofintegrins and immunoglobulin superfamily members (reviewed in Springer,1995; Frenette, 1996; Vestweber, 1999). The selectin family consists ofthree members containing a functional calcium-binding lectin domain. Twoselectins are expressed by endothelial cells (P- and E-selectins) andone is found on most leukocytes (L-selectin) (Kansas, 1996). Geneticanalyses using knockout experiments have shown distinct functions foreach selectin (Frenette, 1997; Robinson et al., 1999). While micelacking a single selectin gene have mildly aberrant phenotypes, animalsdeficient in both endothelial selectins (P/E−/−) show virtually noleukocyte rolling even after cytokine-induced (tumor necrosis factoralpha; “TNF-α”) inflammation (Frenette et al., 1996; Bullard et al.,1996). The profound defect in leukocyte adhesion and extravasation inP/E−/− mice, reminiscent of mice lacking all β₂ integrins, such as micewhich are CD18−/− (Wilson et al., 1993; Scharffetter-Kochanek et al.,1998) suggested that overlapping function of the two endothelialselectins is as important for leukocyte adhesion in vivo as are β₂integrins. In addition to four β₂ integrins (α_(L)β₂(LFA-1), α_(M)β₂(Mac-1), α_(χ)β₂ and α_(D)β₂) leukocytes express other integrins such asα_(V)β₃ and β₁ (on lymphocytes and monocytes but not neutrophils;Carlos, 1994).

[0025] Accordingly, the present invention provides for methods ofdecreasing vaso-occlusion associated with sickle cell disease byinhibiting SS-RBC/leukocyte/endothelial adhesion along any one orseveral steps in the adhesion process.

[0026] Such methods may, for example, but not by way of limitation,inhibit the binding between leukocytes and endothelial P- and/orE-selectin or the binding of leukocyte L-selectin to the endothelium.Such binding may be inhibited, for example, using an immunoglobulinspecific for a selectin molecule, such as a P-, E-, and/or L-selectinmolecule, or a fragment or derivative of such immunoglobulin.Alternatively, such binding may be inhibited using a non-immunoglobulinmolecule which interacts with the calcium-binding lectin domain of theselectin molecule, including molecules which interfere with calciumbinding to the site.

[0027] In other non-limiting embodiments, vaso-occlusion in a sicklecell patient may be decreased by inhibiting the interaction ofleukocytes or RBC/leukocyte complexes with cytokines on the surface ofactivated endothelium. As a non-limiting specific example, an agentwhich inhibits TNF-α, such as an anti-TNF immunoglobulin, fragment orderivative thereof, may be administered.

[0028] In further non-limiting embodiments, vaso-occlusion in a sicklecell patient may be decreased by inhibiting the binding between one ormore elements selected from the group consisting of leukocytes,SS-RBC/leukocyte complexes, and endothelial cells, via a β₂ integrinmolecule. Thus, binding (i) among leukocytes, or (ii) amongSS-RBC/leukocyte complexes, or (iii) between a SS-RBC/leukocyte complexand a leukocyte, or (iv) between an endothelial cell and aSS-RBC/leukocyte complex, or (v) between an endothelial cell and aleukocyte, may be inhibited, for example, by an agent which interfereswith binding of a β₂ integrin molecule, where a β₂ integrin moleculeparticipates, directly or indirectly, in the binding between partners.For example, a leukocyte may be bound to another leukocyte indirectly bybinding to an endothelial cell, and an endothelial cell may be bound toanother endothelial cell indirectly via a plurality of adherentSS-RBC/leukocyte complexes.

[0029] In specific non-limiting examples, binding to α_(L)β₂(LFA-1),α_(M)β₂ (Mac-1), α_(χ)β₂, and/or α_(D)β₂ may be inhibited. Suchinhibition may be achieved, for example, using an immunoglobulinmolecule, or a fragment or derivative thereof, which specifically bindsto the integrin.

[0030] In related embodiments, vaso-occlusion in a sickle cell patientmay be decreased by inhibiting the change in the conformation of β₂integrins into a high-affinity state. Such inhibition may be effected byan immunoglobulin molecule, fragment or derivative thereof or by a smallnon-immunoglobulin molecule.

[0031] In additional non-limiting embodiments, vaso-occlusion in asickle cell patient may be decreased by inhibiting binding among orbetween elements selected from the group consisting of an endothelialcell, a platelet, a leukocyte, and a SS-RBC/leukocyte complex byinhibiting binding via a β₃ integrin, for example, α_(IIb)β₃ or α_(V)β₃integrin. By inhibiting binding via a β₃ integrin, binding between anendothelial cell and either a leukocyte, or a SS-RBC/leukocyte complex,or a platelet, or a SS-RBC/leukocyte/platelet complex, or aplatelet/SS-RBC complex, may be inhibited. Such inhibition may beachieved, for example, using an immunoglobulin molecule or a fragment orderivative thereof which binds to a β₃ integrin. Non-limiting examplesof antibodies which bind to α_(V)β₃ integrin include the murinemonoclonal antibody 7E3 (deposited with the American Type CultureCollection at ATCC HB 8832), the humanized chimeric equivalent of 7E3,c7E3, the Fab fragment of c7E3 (which is sold commercially as ReoPro®),and the monoclonal antibody LM609 and chimeric equivalents. 7E3, c7E3,and Fab 7E3 also bind to α_(IIb)β₃. Where c7E3 or ReoPro is used, thedosage may be, in specific non-limiting embodiments, between 0.1-0.3mg/kg, and preferably 0.25 mg/kg. Preferably, after 0.25 mg/kg isadministered, the patient may further receive intravenous infusion of0.125 m/kg/min for a therapeutically effective period of time.

[0032] In further non-limiting embodiments, vaso-occlusion in a sicklecell patient may be decreased by inhibiting binding between one or moreelements selected from the group consisting of leukocytes,SS-RBC/leukocyte complexes, and endothelial cells, via β₁ integrins.Such inhibition may be achieved using an immunoglobulin molecule, or afragment or derivative thereof, which specifically binds to the β₁integrin.

[0033] In further non-limiting embodiments, vaso-occlusion in a sicklecell patient may be decreased by inhibiting the binding of leukocytes orSS-RBC/leukocyte complexes to von Willebrand factor (vWf). Suchinhibition may be achieved using an immunoglobulin molecule, or afragment or derivative thereof, which specifically binds to the vWf.

[0034] In further non-limiting embodiments, vaso-occlusion in a sicklecell patient may be decreased by inhibiting the binding of leukocytes orSS-RBC/leukocyte complexes to thrombospondin. Such inhibition may beachieved using an immunoglobulin molecule, or a fragment or derivativethereof, which specifically binds to the thrombospondin.

[0035] In further non-limiting embodiments, vaso-occlusion in a sicklecell patient may be decreased by inhibiting the binding of leukocytes orRBC/leukocyte complexes to a molecule, such as, butnotlimitedto, ICAM-1,VCAM-1, ortheirligands CD18 and α₄β₁. Such inhibition may be achievedusing an immunoglobulin molecule, or a fragment or derivative thereof,which specifically binds to the endothelial adhesion molecule.

Methods of Identifying Agents Useful in Treating Sickle Cell Disease

[0036] The present invention provides for methods of identifying agentsuseful in treating sickle cell disease which comprise determiningwhether a test agent is able to modulate the adhesion of SS-RBC toleukocytes and thereby to venular endothelium. Such methods may bepracticed in vitro or in vivo. Examples of in vitro studies may includeassays which test for SS-RBC/leukocyte binding by, for example,co-precipitation or co-sedimentation, or by retention on a solid matrix.

[0037] Alternatively, the effectiveness of the test agent at inhibitingadhesion may be evaluated in vivo. For example, but not by way oflimitation, the test agent may be evaluated using intravital microscopy,using techniques as set forth in Example Section 6 below.

[0038] The ability of a test agent to inhibit the binding of a SS-RBC toa leukocyte, and/or inhibit the binding of a SS-RBC/leukocyte complex ora leukocyte to an endothelium or to endothelial cells, indicates thatthe test agent may be useful in the treatment of sickle cell disease. Incertain although not all circumstances, it may be desirable to determinethat the test agent selectively blocks adhesion of sickled rather thannon-sickled erythrocytes; in such circumstances, the amount of availableoxygen may be decreased or increased to maximize or minimize,respectively, the formation of SS-RBC.

[0039] Because many of the molecules involved in the adhesion pathwayare important to normal biological function, it may be desirable toselect for agents which have a short half life for administration duringsickle cell crises, or which change conformation and become more activeat lower oxygen tensions.

Animal Model Systems

[0040] The present invention further provides for animal model systemswhich are designed to lack one or more element of the adhesion pathway,including, for example, those elements set forth in Section 5.1, supra.Such animals may be transgenic animals, including, but not limited to,transgenic mice, lacking or, alternatively, overexpressing a geneencoding a protein selected from the group consisting of a selectin,such as P-, E- or L-selectin; a chemokine, such as TNF-α; a β₂ integrin,such as α_(L)β₂(LFA-1), α_(M)β₂ (Mac-1), α_(χ)β₂, and α_(D)β₂; a β₃integrin, for example, α_(V)β₃; a β₁ integrin; vWf, thombospondin,ICAM-1, VCAM-1, CD18 and α₄β₁.

EXAMPLE: SICKLE CELL INTERACTIONS WITH ADHERENT LEUKOCYTES CAN INITIATEVENULAR OCCLUSION IN SICKLE CELL MICE

[0041] Materials and Methods. Sickle cell breeding pairs were obtainedfrom Dr. Mohandas Narla at the Lawrence Berkeley Institute, and weremaintained according to Dr. Narla's instructions. “Heterozygotes”,referred to herein as “SA” mice, express the sickle transgene, aredeficient in α globin and heterozygous for the β-globin locus, and aregenetically Tg(Hu-miniLCRα1^(G)γ^(A)γδβ^(S))mα−/−β−/+. Female SA micewere bred with male sickle cell mice, which express exclusively humanglobins, and are genetically Tg(Hu-miniLCRα1^(G)γ^(A)γδβ^(S))mα−/−β−/−.After the progeny from these breedings were weaned, a drop of blood wasobtained from a tail biopsy to permit phenotyping by hemoglobinelectrophoresis. To generate large numbers of male sickle cell mice, abone marrow transplantation strategy was used which aimed atreconstituting the entire blood compartment of several recipient micefrom precursors obtained from a single sickle cell mouse. Fresh femoralbone marrow cells were obtained from one female sickle cell and one“heterozygous” control mouse (derived from the same genetic backgroundas the sickle animals; Paszty, 1997). Wild-type male C57B1/6 recipientmice were lethally irradiated with 1200 cGy, in two split doses, andinjected, under a sterile hood, with bone marrow nucleated cells from SSor SA animals, at a dose of 1.5×10⁶ cells per recipient. Following theprocedure, transplanted animals were transferred into a sterile cagecontaining sterile food and water (see Frenette, 1998, Frenette, 2000).Since the life-span of the normal mouse RBC is approximately 55 days(Hoffman-Fezer, 1993), mice were allowed to recover for at least twomonths prior to evaluation for engraftment and chimerism.

[0042] Between 8 and 12 weeks after transplantation, blood was obtainedfrom a small tail incision and hemoglobin was separated on apolyacrylamide gel containing urea and Triton-X-100 (Alter et al.,1980); the results are shown in FIG. 2. 10 micrograms of protein,determined spectrophotometrically, were loaded per lane. The first twolanes represent normal mouse and human hemoglobin and the next fivelanes represent mixtures with decreasing amounts of human hemoglobin.Under these electrophoretic conditions, the mouse and human β globinsco-migrate; however, the mouse and human α globin can be easilydistinguished. The various hemoglobin mixtures demonstrate that theassay can detect as little as 1-2% human hemoglobin. The second half ofthe gel are samples from representative animals transplanted with SSbone marrow, and shows that the RBCs from four wild-type (“WT”)recipients and three P-/E- −/− mice (see Section 7, infra) contained>97%human globins. In addition, wild-type animals transplanted with SS bonemarrow cells (hereafter referred to as SS-WT) were severely anemic anddisplayed very significant splenomegaly, compared with animals thatreceived SA bone marrow (SA-WT). Thus, these results indicate that theSS phenotype can be transplanted into adult wild-type recipient mice.

[0043] The cremasteric microcirculation of the highly chimeric animalswas evaluated using intravital microscopy. The surgical preparation ofthe cremaster muscle itself induces inflammatory stimuli leading toleukocyte rolling and progressive recruitment of adherent leukocytes.More severe inflammation may be induced by administering TNF-α, aninflammatory cytokine which induces P- and E-selectin-mediated leukocyterolling (Frenette, 1996; Bullard, 1996). Because inflammation isclinically known to trigger sickle cell crises, chimeric mice weretreated with murine recombinant TNF-α (0.5 micrograms intrascrotally)3.5 hours prior to preparing the cremaster muscle for intravitalmicroscopy (Frenette, 1996; Frenette, 1998; Ley, 1995; Bullard, 1996).While treatment with TNF-α was tolerated well in SA-WT controls, SS-WTmice died during or soon after surgery. However, SS transplants survivedthe surgery when not pre-treated with the cytokine or when treated witha half dose of TNF-α (however, the half-dose did not produce meaningfulinflammation, as assessed by the lack of leukocyte rolling velocities).The following protocol was therefore designed to induce a progressiveinflammatory response in SS and SA transplants (FIG. 3).

[0044] As depicted diagrammatically in FIG. 3, mice were prepared forthe cremasteric intravital microscopy using standard procedures(Pemberton, 1993; Kaul, personal communication). Mice were anesthetizedwith urethane/chlorose and a tracheostomy was made to facilitatespontaneous respiration. Immediately after the cremaster dissection, theanimal was placed on a plexiglass stage and the cremaster muscle wascontinuously perfused with an endotoxin-free bicarbonated solution (NaCl135 mM, KCl 5 mM, NaHCO₃ 27 mM, MgCl₂ 0.64 mM) equilibrated with 95%N₂/5% CO₂ at 37° C. The tissue was allowed to stabilize for 15 minutes,at which point microvessels (post-capillary and collecting venules) werevideotaped until 90 minutes after surgery (IVM-1; FIG. 3). At the 90minute time point, TNF-α (0.5 micrograms, intraperitoneally) wasinjected and allowed to take effect for 90 minutes, and microvesselswere recorded for 90 minutes (IVM-2; FIG. 3). When possible,approximately 7-10 venules were recorded before and after TNF-αadministration in each experiment. Prior to timing each vessel,centerline RBC velocities were measured in real time using an opticaldoppler velocimeter. Vessel diameter and shear rates were determined aspreviously described (Frenette, 1996).

[0045] Results and Conclusions. Vaso-occlusion is a major cause ofmorbidity and mortality in sickle cell disease. To better understand thepathophysiology of vaso-occlusion in vivo, intravital microscopy wasperformed in (1) C57B1/6 wild-type (“WT”) mice; (2) mice exclusivelyexpressing sickle cell hemoglobin (“SS”;[Tg(Hu-miniLCRα1^(G)γ^(A)γδβ^(S))mα−/−β−/−]); and (3) lethallyirradiated WT mice transplanted with bone marrow from either SS mice ormice heterozygous for sickle hemoglobin (“SA”;human β^(S)/mouseβ[Tg(Hu-miniLCRα1^(G)γ^(A)γδβ^(S))mα−/−β−/+].

[0046] In the transplant recipients, three months after transplantation,SS bone marrow recipients had >96% donor hemoglobin and displayed severeanemia (hematocrit 21±3%; n=10, p<0.05), high reticulocyte counts andsplenomegaly; by comparison, heterozygous bone marrow recipients hadnearly normal hematocrits (32±3%; n=7) and only a slight increase inspleen weight (ratio: 5±1).

[0047] The cremasteric muscle of the mice was then surgically dissected,and “small” (15-20 μm) and “large” (30-50 μm) venules were visualizedbetween 15-90 minutes after surgery and after treatment with TumorNecrosis Factor a (“TNF-α”; 0.5 μg/mouse). The surgery itself producedan inflammatory response leading to leukocyte adhesion, and thisresponse was accentuated by TNF-α treatment.

[0048] Although occasionally direct interaction between SS-RBCs and thevasculature was observed, the most striking finding was that numerousSS-RBCs interacted with adherent leukocytes in venules activated bysurgery alone and these interactions were increased after TNF αadministration. On average, 17±5 SS-RBCs interacted with adherentleukocytes per minute over 100 μm venular length in SS-BMT mice (n=34venules in 5 mice) (FIG. 4A). These interactions began approximately 30minutes after the surgery and continued throughout the observationperiod (FIG. 4B). Similar interactions were seen in non-transplanted SSmice. Very few SS-RBC/leukocyte interactions were observed in SA-BMTanimals (0.04±0.03/min/100 μm; n=24 venules in 4 mice) and none wereseen in wild-type animals. The graph in FIG. 4B shows that there wererelatively few RBC/leukocyte interactions in the first half of the IVM-1period, and that the number of interactions drastically increased duringthe second half of filming (r=0.41, p=0.005).The tethers resisted theshear stress of the flowing blood and lasted up to 100 seconds. In smallvenules, SS-RBCs formed transient bridges between adherent leukocytesand between adherent leukocytes and the endothelium, resulting inobstruction of blood flow which could be either transient or prolonged.Following TNF-α stimulation, continuous SS-RBC/leukocyte adhesion eventslead to a significant decrease in blood flow in SS-BMT mice compared toSA-BMT animals (shear rates: 501±35 versus 110±29; n=30-32; p<0.0001).

[0049] FIGS. 5A-C illustrate examples of digital still frames obtainedfrom representative video recordings. FIG. 5A shows a venule stimulatedby surgery followed by TNF-α treatment. Rolling and adherent leukocytesare present but RBC adhesive interactions are rare (none were seen inthis mouse). In FIG. 5B, numerous SS-RBCs (elongated cells, whitearrowheads) are seen to interact with adherent leukocytes (arrows).Consistent with a true adhesive interaction (as compared to physicaltrapping), RBC/leukocyte interactions can resist the shear of venulesfor several seconds. This is particularly evident in FIG. 5C, an imagetaken of a large venule (approx. 90 microns) where two SS-RBCs, onesickled in shape (arrowhead) the other with a normal discoid shape,remained bound to an adherent leukocyte, resisting the shear of flowingblood. Although most interacting RBCs appear to be sickle-shaped, normaldiscoid cells were occasionally seen, as in FIG. 5C. This suggests that,unlike sickle/endothelium adhesion, the cell density profile may notplay an important role in the interaction with adherent leukocytes (orthe high-density cells may be more adherent to leukocytes). It shouldalso be noted that in small venules (<20 microns), the interaction offew RBCs (or only one RBC in the smallest venule) with one adherentleukocyte could transiently (or permanently) occlude blood flow.

[0050] After TNF-α, RBC/leukocyte interactions increased (or persisted)in SS-WT mice (but were not increased in SA-WT animals) and lead to aprogressive reduction in blood flow in the cremaster microvasculature.FIG. 6 illustrates the shear rates before and after TNF-α administrationin SA and SS transplants. Shear rates are directly proportional to themean RBC velocity and inversely proportional to the vessel diameter.While shear rates between SA and SS transplants were similar beforeTNF-α, shear rates were significantly reduced after TNF-α in SS mice(approximately 80 percent reduction) compared to SA mice. Moreover, fourout of the five studied SS transplanted mice died during the recordingafter TNF-α administration whereas there was no lethality in the SA-WTgroup. Since TNF-α increases the number of adherent leukocytes invenules (Morita, 1995; Ley, 1995), these results suggest that TNF-αadministration to SS transplanted mice leads to a severe (often lethal)vaso-occlusive crisis. However, it was also possible that TNF-α mightproduce other lethal effects in SS-WT mice that are independent ofleukocyte adhesion.

[0051] These observations suggest a critical role for SS-RBC/leukocyteinteractions in initiating vaso-occlusive episodes in sickle cell mice.They are in accord with the documented correlation between low leukocytecounts and reduced painful crises in hydroxyurea-treated patients aswell as in in vitro studies of SS-RBC/leukocyte interactions by Hofstraet al.

EXAMPLE: P- AND E-SELECTIN DEFICIENCY PROTECTS AGAINST TNF-α INDUCEDVASCULAR OCCLUSION IN SICKLE MICE: EVIDENCE FOR A CRITICAL ROLE FORADHERENT LEUKOCYTES

[0052] To further evaluate the role of adherent leukocytes in sicklecell disease, bone marrow from mice exclusively expressing sickle cellhemoglobin (“SS”;[Tg(Hu-miniLCRα1^(G)γ^(A)γδβ^(S))mα−/−β−/−]) wastransplanted into mice lacking both P- and E-selectins (P/E−/−). P/E−/−mice have severe defects in leukocyte rolling and adhesion in inflamedvenules. Experimental data (FIGS. 7A-B) indicates that the amount ofleukocyte rolling and adhesion and the blood flow was preserved evenafter TNF-α stimulation (shear rates: 604±57, n=29) (FIGS. 8A-B). Theinteractions per adherent leukocyte were not, however, altered.

[0053] Consistent with reduced numbers of adherent leukocytes in SS-P/E−/− mice, the total number of erythrocyte/leukocyte interactions wassignificantly reduced in P/E−/− mice harboring SS-RBCs (0.4±0.3/min/100μm; n=23 in 3 mice; p=0.01). It is interesting to note that theremaining adherent leukocytes present in endothelial selectin-deficientvenules could still interact with SS-RBCs, suggesting that P/E selectinsare not necessary for SS-RBC/leukocyte interactions.

[0054] Unlike SS-WT mice which for the most part died during theintravital experiment, all SS-P/E−/− mice survived the entireexperiment. These results strongly support a role for adherentleukocytes in initiating vasoconstriction by interacting withcirculating sickle erythrocytes, and indicate that P- and E-selectindeficiencies protect SS mice from vaso-occlusion. Moreover, the datasuggest that the absolute number of interacting leukocytes in a givenvenule, rather than the rate of interactions per leukocyte, appear to bea critical factor in venular occlusion.

[0055] Determination of blood counts and assessment of spleenweight/body weight ratios among various transplanted andnon-transplanted groups revealed several abnormalities. The preliminaryblood counts were done after TNF-α treatment, except 4 SS bone marrowdonor mice (Table 1, 4th row), which were performed at baselineconditions. In addition to being severely anemic, these resting SS miceexhibited severe leukocytosis, in contrast to WT or SA mice which showeda mild leukocytosis after TNF-α (normal WT WBC counts are ˜3 to5×10³/μl). WBCs were lower after TNF-α administration in SS mice,possibly resulting from increased adhesion to the vessel wall duringinflammation. Both SS-WT and SA-WT chimeras displayed blood countssimilar to their non-transplanted donor counterparts, suggesting thatthis transplantation model reproduced very well the phenotype of sicklecell mice. TABLE 1 Blood counts and spleen weights of intact andtransplanted mice Leukocytes × Platelets (× Spleen wgt. ratio TNF-α10³/μl Hematocrit (%) 10⁶/μl) (g/g BW × 10⁻³) WT (n = 3) Yes 7.0 ± 1.343.9 ± 6.9 1009 ± 326 3.0 ± 0.4 SA mice (n = 6) Yes 10.7 ± 2.4  29.8 ±5.6 1500 ± 162 5.0 ± 0.3 SA

WT (n = 4) Yes 6.3 ± 0.3 35.1 ± 0.6 1008 ± 32  3.0 ± 0.1 SS mice (n = 4)No 46.1 ± 9.1  19.7 ± 1.5  636 ± 108 N/E SS mice (n = 4) Yes 14.3 ± 5.3 12.6 ± 2.5 288 ± 52 53 ± 1  SS

WT (n = 4) Yes 23.0 ± 2.9  11.2 ± 0.6 272 ± 19 28 ± 2  SS

P/E-/- (n = 3) Yes 84.7 ± 14.9 21.7 ± 5.1 233 ± 62 39 ± 5 

[0056] Leukocytosis was also more severe in P/E−/− mice expressing SShemoglobin. Interestingly, the blood from TNF-α treated SS mice (andfrom the chimeras generated by transplantation) contained much fewerplatelets suggesting platelet consumption during the vaso-occlusiveprocess. Although this might suggest a role for platelets invaso-occlusion, the fact that a similar reduction in platelet numbers isseen in SS

P/E−/− mice (and that SS

P/E−/− mice are protected) argues that platelets may not be necessaryfor vaso-occlusion. The lower spleen weight in transplanted micecompared to their non-transplanted controls likely results from the factthat transplanted animals have had sickle cell disease for only a fewweeks.

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[0199] Various publications are cited herein, the contents of which arehereby incorporated by reference in their entireties.

What is claimed is:
 1. A method of treating sickle cell diseasecomprising administering, to a subject in need of such treatment, atherapeutic amount of an agent which decreases venular occlusion bysickle erythrocytes adherent to leukocytes by inhibiting binding betweenelements selected from the group consisting of an endothelial cell, aplatelet, a leukocyte, and a sickle erythrocyte/leukocyte complex. 2.The method of claim 1 wherein venular occlusion is decreased byinhibiting a process selected from the group consisting of the adherenceof sickle erythrocytes to leukocytes, the adherence of leukocytes to thevenule endothelium, and the adherence of a sickle erythrocyte/leukocytecomplex to the venule endothelium.
 3. The method of claim 1, whichcomprises administering, to the subject, an agent which inhibits thebinding of a leukocyte via a selectin molecule.
 4. The method of claim3, wherein the selectin molecule is selected from the group consistingof P-selectin, E-selectin, and L-selectin.
 5. The method of claim 1,which comprises administering, to the subject, an agent which inhibits acytokine.
 6. The method of claim 5, wherein the cytokine is tumornecrosis factor alpha.
 7. The method of claim 1, which comprisesadministering, to the subject, an agent which inhibits the bindingbetween one or more elements selected from the group consisting ofleukocytes, sickle erythrocyte/leukocyte complexes, and endothelialcells, via a β₂ integrin molecule.
 8. The method of claim 7, wherein theβ₂ integrin molecule is selected from the group consisting ofα_(L)β₂(LFA-1), α_(M)β₂ (Mac-1), α_(χ)β₂, and α_(D)β₂.
 9. The method ofclaim 1, which comprises administering, to the subject, an agent whichinhibits a change in the conformation of β₂ integrins into ahigh-affinity state.
 10. The method of claim 1, which comprisesadministering, to the subject, an agent which inhibits binding betweenelements selected from the group consisting of endothelial cells,platelets, leukocytes and sickle erythrocyte/leukocyte complexes byinhibiting binding via a β₃ integrin.
 11. The method of claim 10,wherein the β₃ integrin is selected from the group consisting ofα_(IIb)β₃ and α_(V)β₃ integrin.
 12. The method of claim 11, wherein theagent is selected from the group consisting of murine monoclonalantibody 7E3, as deposited with the American Type Culture Collection andassigned accession number ATCC HB 8832, the humanized chimericequivalent of 7E3 which is c7E3, the Fab fragment of c7E3, themonoclonal antibody LM609 and a humanized version thereof.
 13. Themethod of claim 1, which comprises administering, to the subject, anagent which inhibits binding between one or more elements selected fromthe group consisting of leukocytes, sickle erythrocyte/leukocytecomplexes, and endothelial cells, via a β₁ integrin molecule.
 14. Themethod of claim 1, which comprises administering, to the subject, anagent which inhibits the binding of a leukocyte or a sickleerythrocyte/leukocyte complex to von Willebrand factor.
 15. The methodof claim 1, which comprises administering, to the subject, an agentwhich inhibits the binding of a leukocyte or a sickleerythrocyte/leukocyte complex to thrombospondin.
 16. The method of claim1, which comprises administering, to the subject, an agent whichinhibits the binding of a leukocyte or a sickle erythrocyte/leukocytecomplex to a molecule selected from the group consisting of ICAM-1 andVCAM-1
 17. A method for identifying an agent useful in treating sicklecell disease comprising: (i) contacting SS-RBCs and leukocytes in thepresence of a test agent; (ii) comparing the binding of SS-RBCs andleukocytes in the presence of a test agent to the level of binding inthe absence of a test agent wherein a decrease in adhesion of SS-RBC toleukocytes in the presence of the test agent as compared to the adhesionin the absence of the test agent indicates the identification of anagent useful in treating sickle cell disease.
 18. The method of claim 17wherein the adhesion of SS-RBCs to leukocytes is detected byco-precipitation assays.
 19. The method of claim 17 wherein the adhesionof SS-RBCs to leukocytes is detected by co-sedimentation assays.
 20. Themethod of claim 17 wherein the adhesion of SS-RBCs to leukocytes isdetected by co-retention on a solid matrix.
 21. The method of claim 17further comprising determining whether the agent inhibits adhesion ofnon-sickled RBCs to leukocytes.
 22. The method of claim 17 wherein theadhesion of RBCs to leukocytes is measured using intravital microscopy.